The Program Project focuses on three major aspects of human prion diseases: 1) structure of human PrPSc, 2) non-invasive, accurate diagnosis;3) prion diversity and phenotypic determination, and includes three Research Projects. Aims 1-3 of Project 1 will determine the conformational structure of human PrP amyloid and will use recombinant PrP as substrate to generate recombinant "PrPSc" by protein misfolding cyclic amplification (PMCA) in collaboration with Research Project 2. Infectivity and conformational structure of recombinant PrPSc will be assessed. In Aim 1, Project 2 will apply PMCA to develop a novel approach to the diagnosis of human prion diseases by attempting to detect PrPSc likely to be present in blood, urine, cerebrospinal fluid and skeletal muscle of patients with prion diseases. In Aim 2, carried out in collaboration with Project 3, PMCA will be used to study the conversion of human PrPc triggered by PrPSc from different animal species using human PrPc as substrate, thus "humanizing" the replicated animal PrPSc. In vitro replication by PMCA may overcome the species barrier encountered by some animal PrPSc. In Aims 3 and 4, PMCA will also be used to assess the competence to convert PrPc of i) unconventional forms of human PrPSc difficult to replicate by bioassay and ii) PrPSc forms in different states of aggregation. Aims 1 and 2 of Project 3 will use i) transgenic mice expressing human PrP [Tg(HuPrP)] that is glycosylation-impaired and ii) and Tg(HuPrP) mice inoculated with PrPSc species in different states of aggregation to investigate how glycans and aggregation respectively, influence infectivity and expression of the disease phenotype. Aim 3 will take advantage of PMCA technology to assess the role of PrP genotype at codon 129 in specifying prion strain characteristics. Aim 4 will determine whether the animal PrPSc replicated by PMCA with human PrPc as substrate in Project 2 transmits the disease to Tg(HuPrP) mice thus proving the PMCA capacity of circumventing species barriers. Transmissibility to Tg(HuPrP) mice of bovine amyloidotic spongiform encephalopathy (BASE), a novel prion disease, will also be assessed. Aim 5 is dedicated to the detailed characterization of a novel human prion disease associated with protease-sensitive PrPSc and lacking mutation in the PrP gene coding region despite family history of dementia. Administrative, Animal and Tissue Cores are proposed that will provide well characterized animals, animal and human tissues, and body fluids. The Program Project takes advantage of the diverse but complementary expertise of three senior investigators to propose highly interactive studies likely to significantly advance research and diagnosis in prion diseases.